The major objective of this grant proposal is to understand how HIV-1 infects cells. Entry of HIV-1 into target cells requires the binding of the virus to its cellular receptor, the CD4 glycoprotein, followed by fusion of the virion and cellular membranes and release of the viral core into the cytoplasm. Both the binding and fusion processes have been shown to be mediated by the envelope glycoprotein. Considerable details on the initial interaction of envelope gpl20 and CD4 have been reported, but the subsequent steps leading to membrane fusion are still poorly understood. Recent data indicate that the V3 hypervariable region of gpl2O is involved in these post-binding processes, and that sequences within this domain determine HIV-1 infectivity, host range and syncytium-forming ability. However, other regions of the envelope glycoprotein have also been found to affect these biological properties of HIV-1. Furthermore, the mechanism by which the V3 loop mediates viral entry, and what other cellular component(s) interacts with V3 in determining viral entry and tropism are still unknown. Three specific aims are proposed to address these questions. First, with the use of recombinant and mutant viruses, we will continue our structure/function analyses of the HIV-1 genome to identify functional domains that interact with V3 to determine the replicative, host range and cytopathic properties of HIV-1. Second, we will test the hypothesis that the amino acid composition of the V3 loop influences the conformational changes in envelope glycoprotein that are induced by gpl2O/CD4 binding. We will monitor these conformational changes by determining the differential binding of MAb to wild-type and mutant virion or cell surface gpl2O, the differences in proteolytic digestion of wild-type and mutant virion gpl2O, and the differences in sensitivity to serum neutralization of wild-type and mutant viruses. Finally, by gene transfer experiments, we will attempt to identify the accessory cellular factor(s) that appears to be required for viral entry. An understanding of the mechanism by which HIV-1 infects cells, and the cellular factor(s) involved, may lead to the identification of new targets for anti-viral and vaccine developments.